Senescent cell biomarkers

ABSTRACT

The invention relates to senescent cell biomarkers and the uses thereof. The invention also extends to methods and kits for detecting senescence, and drug conjugates and pharmaceutical compositions for killing senescent cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the U.S. national phase of International Application No.PCT/GB2015/051480, filed May 20, 2015, which claims the benefit ofUnited Kingdom Patent Application No. 1409519.4, filed May 29, 2014,which are incorporated by reference in their entirety.

The present invention relates to senescent cells, and in particular, tobiomarkers that are assembled into, or associated with, the plasmamembrane of senescent cells, and to methods of identifying senescentcells. The invention also extends to therapies, compositions and methodsfor targeting and killing senescent cells, and uses thereof.

Apoptosis and senescence have been proposed to be the two main processesthat prevent the emergence of transformed cells. Senescence is definedas a permanent cell cycle arrest in which cells remain metabolicallyactive and adopt characteristic phenotypic changes. Senescent cellsoften appear multinucleated, large and extended, and exhibit spindle andvacuolisation features. The establishment of this phenotype is believedto be either the result of telomere shortening after a number of celldivisions (replicative senescence) or a response to stress stimuli(stress-induced premature senescence, SIPS). Expression of oncogenes,such as Ras, cyclin E, E2F3 and Raf can also trigger senescence, whichsupports the tumour suppressing properties of senescence.

The presence of senescent cells in vivo is often observed in thepre-malignant stages of a tumour and they progressively disappear,suggesting that the senescent barrier needs to be overcome in order toprogress into full malignancy. Moreover, cell senescence has long beenassociated with age-dependent organismal changes, since accumulation ofsenescent cells has been shown to contribute to the functionalimpairment of different organs. This has led to the hypothesis thatsenescence is an antagonistically pleiotropic process, with beneficialeffects in the early decades of life of the organism as a tumoursuppressor but that can be detrimental to fitness and survival in laterstages due to its involvement in ageing.

One of the well-known features of both replicative and stress-inducedsenescence is the participation of the p53-p21 and/or p16-RB pathways.In vivo suppression of p53, and/or its upstream regulator ARF, is enoughto prevent senescence in some models. However, other cell types relyprimarily on p16 for senescence induction. The p53 target gene, p21, hasoften been considered critical for establishing senescence, whereas p16could be more involved in the maintenance of the phenotype, togetherwith an increase in intracellular Reactive Oxygen Species (ROS). Othergenes upregulated in senescent cells are PPP1A, Smurf2 and PGM.

Cellular senescence is also associated with the secretion of growthfactors, chemokines and cytokines, known as the senescence-associatedsecretary phenotype (SASP). SASPs have shown an effect on cellproliferation and angiogenesis, as well as in promoting ageing. Also,SASP can induce migration of leukocytes and tumour cells, which in turnmay induce tumour metastasis. Increased expression of intracellularand/or secreted proteins has often been used as a surrogate marker ofsenescence, although it is not a specific measurement.

Senescent cells also display different modifications in the organisationof chromatin that can help identify them. In normal cells, DNA stainingreveals completely uniform colour outlines, whereas senescent cellsusually show dot-like patterns, known as senescence-associatedheterochromatic foci (SAHF). This phenomenon is due to intensiveremodelling in the chromatin, which results in less susceptibility fordigestion by nucleases. SAHF development is not necessary for senescenceto occur, and this depends primarily on cell types and senescentstimuli.

Apart from all this, the most distinctive measurable feature ofsenescent cells is the presence of β-galactosidase enzymatic activity.This enzyme normally displays activity at pH 4.0 within lysosomes, butin senescent cells it is also active at pH 6.0. This phenomenon istermed senescence associated-β-galactosidase (SA-β-Gal). Although thereasons for this are not completely clear, it is thought to be due to anenlargement in the structure of lysosomes in senescent cells. SA-β-Galhas not been shown to have any role in the establishment or maintenanceof the senescent phenotype. Although it is currently the standard forthe detection of senescent cells, high cell confluence and treatmentwith hydrogen peroxide can also stimulate SA-β-Gal activity, leading tomany false positives. None of the currently available markerssatisfactorily or conclusively identify senescent cells in vivo or invitro, which underscores the need for better characterization tools.

Despite the considerable knowledge accumulated in the fifty years sinceLeonard Hayflick first described the phenomenon of senescence, themolecular pathways involved in the establishment and maintenance of thesenescent phenotype still have not been fully characterized. Forinstance, little is known about the profile of proteins specificallyexpressed in the membrane of senescent cells, which could be criticalfor the immune clearance of senescent cells observed in certainsituations.

There is therefore a need for more specific and sensitive senescent cellbiomarkers.

The inventors have studied the expression profile of plasma membraneproteins in senescent cells in order to identify novel markers thatcould be easily recognized and propose potential effectors andmodulators of the senescent pathway. Ten novel specific markers ofsenescence were validated, and two of these were selected in order todevelop a fast and straightforward FACS-based approach to identifysenescent cells. The results described herein will facilitate the studyof senescent cells and provide new insights on pathways that contributeto this mechanism. In addition, identification of these ten newsenescence cell biomarkers will be very useful in targeting senescentcells for treating conditions associated with cell senescence.

Thus, according to a first aspect of the invention, there is providedthe use of at least one polypeptide selected from DEP-1, NTAL, EBP50,STX4, VAMP3, ARMCX-3, LANCL1, B2MG, PLD3 and VPS26A, or a variant orfragment thereof, as a senescent cell biomarker.

Advantageously, all of the senescent cell biomarkers used in accordancewith the first aspect of the invention are associated with the plasmamembrane of senescent cells. The biomarkers exhibit low or non-existentbasal expression in non-senescent cells and/or are inducible insenescent cells. Consequently, these biomarkers are extremely specificand sensitive to detection. Some of the biomarkers according to theinvention contain at least one domain or epitope, which is exposed onthe extracellular surface of senescent cells (see Table 1), whereas theremaining biomarkers are expressed intracellularly and are associatedwith the plasma membrane of senescent cells. Biomarkers that areexpressed on the surface of senescent cells enable senescent cells to bedetected more quickly and easily compared to the known, and more widelyused senescent cell biomarker, SA-β-Gal, which is considered anunreliable biomarker. Therefore, it will be appreciated that thesenescent biomarkers disclosed herein can be readily detected using avariety of simple, conventional techniques known in the art.

TABLE 1 Subcellular location and membrane topology of biomarkers showingincreased expression in senescent cells Senescent biomarker Subcellularlocation DEP-1 Integral transmembrane protein of plasma membrane NTALIntegral transmembrane protein of plasma membrane EBP50 Peripheralmembrane protein associated with the cytoplasmic face of the plasmamembrane (intracellular) STX4 Membrane anchored protein with a largeintracellular domain and no extracellular domain VAMP3 Membrane anchoredprotein with a large intracellular domain and no extracellular domainARMCX-3 Integral plasma membrane protein with two transmembrane helicesand two extracellular domains LANCL1 Integral plasma membrane proteinwith six transmembrane helices and three extracellular domains B2MGSecreted protein (extracellular) found associated with the extra::cytoplasmic face of the plasma membrane PLD3 Trans membrane protein ofplasma membrane with a single extracellular domain VPS26A Peripheralmembrane protein associated with the cytoplasmic face of the plasmamembrane (intracellular)

DEP-1 is an integral transmembrane membrane protein of the plasmamembrane. The amino acid sequence of DEP-1 (Accession code: Q12913; alsoknown as CD148 or PTPRJ) is referred to herein as SEQ ID No.1, asfollows:

[SEQ ID No. 1] MKPAAREARL PPRSPGLRWA LPLLLLLLRL GQILCAGGTPSPIPDPSVAT VATGENGITQ ISSTAESFHK QNGTGTPQVETNTSEDGESS GANDSLRTPE QGSNGTDGAS QKTPSSTGPSPVFDIKAVSI SPTNVILTWK SNDTAASEYK YVVKHKMENEKTITVVHQPW CNITGLRPAT SYVFSITPGI GNETWGDPRVIKVITEPIPV SDLRVALTGV RKAALSWSNG NGTASCRVLLESIGSHEELT QDSRLQVNIS GLKPGVQYNI NPYLLQSNKTKGDPLGTEGG LDASNTERSR AGSPTAPVHD ESLVGPVDPSSGQQSRDTEV LLVGLEPGTR YNATVYSQAA NGTEGQPQAIEFRTNAIQVF DVTAVNISAT SLTLIWKVSD NESSSNYTYKIHVAGETDSS NLNVSEPRAV IPGLRSSTFY NITVCPVLGDIEGTPGFLQV HTPPVPVSDF RVTVVSTTEI GLAWSSHDAESFQMHITQEG AGNSRVEITT NQSIIIGGLF PGTKYCFEIVPKGPNGTEGA SRTVCNRTVP SAVFDIHVVY VTTTEMWLDWKSPDGASEYV YHLVIESKHG SNHTSTYDKA ITLQGLIPGTLYNITISPEV DHVWGDPNST AQYTRPSNVS NIDVSTNTTAATLSWQNFDD ASPTYSYCLL IEKAGNSSNA TQVVTDIGITDATVTELIPG SSYTVEIFAQ VGDGIKSLEP GRKSFCTDPASMASFDCEVV PKEPALVLKW TCPPGANAGF ELEVSSGAWNNATHLESCSS ENGTEYRTEV TYLNFSTSYN ISITTVSCGKMAAPTRNTCT TGITDPPPPD GSPNITSVSH NSVKVKFSGFEASHGPIKAY AVILTTGEAG HPSADVLKYT YEDFKKGASDTYVTYLIRTE EKGRSQSLSE VLKYEIDVGN ESTTLGYYNGKLEPLGSYRA CVAGFTNITF HPQNKGLIDG AESYVSFSRYSDAVSLPQDP GVICGAVFGC IFGALVIVTV GGFIFWRKKRKDAKNNEVSF SQIKPKKSKL IRVENFEAYF KKQQADSNCGFAEEYEDLKL VGISQPKYAA ELAENRGKNR YNNVLPYDISRVKLSVQTHS TDDYINANYM PGYHSKKDFI ATQGPLPNTLKDFWRMVWEK NVYAIIMLTK CVEQGRTKCE EYWPSKQAQDYGDITVAMTS EIVLPEWTIR DFTVKNIQTS ESHPLRQFHFTSWPDHGVPD TTDLLINFRY LVRDYMKQSP PESPILVHCSAGVGRTGTFI AIDRLIYQIE NENTVDVYGI VYDLRMHRPLMVQTEDQYVF LNQCVLDIVR SQKDSKVDLI YQNTTAMTIY ENLAPVTTFG KTNGYIA

Preferably, the extracellular domain of DEP-1 is used as a biomarker ofsenescent cells. The amino acid sequence of the extracellular domain ofDEP-1 is referred to herein as SEQ ID No. 2, as follows:

[SEQ ID No. 2] AGGTPSPIPD PSVATVATGE NGITQISSTA ESFHKQNGTGTPQVETNTSE DGESSGANDS LRTPEQGSNG TDGASQKTPSSTGPSPVFDI KAVSISPTNV ILTWKSNDTA ASEYKYVVKHKMENEKTITV VHQPWCNITG LRPATSYVFS ITPGIGNETWGDPRVIKVIT EPIPVSDLRV ALTGVRKAAL SWSNGNGTASCRVLLESIGS HEELTQDSRL QVNISGLKPG VQYNINPYLLQSNKTKGDPL GTEGGLDASN TERSRAGSPT APVHDESLVGPVDPSSGQQS RDTEVLLVGL EPGTRYNATV YSQAANGTEGQPQAIEFRTN AIQVFDVTAV NISATSLTLI WKVSDNESSSNYTYKIHVAG ETDSSNLNVS EPRAVIPGLR SSTFYNITVCPVLGDIEGTP GFLQVHTPPV PVSDFRVTVV STTEIGLAWSSHDAESFQMH ITQEGAGNSR VEITTNQSII IGGLFPGTKYCFEIVPKGPN GTEGASRTVC NRTVPSAVFD IHVVYVTTTEMWLDWKSPDG ASEYVYHLVI ESKHGSNHTS TYDKAITLQGLIPGTLYNIT ISPEVDHVWG DPNSTAQYTR PSNVSNIDVSTNTTAATLSW QNFDDASPTY SYCLLIEKAG NSSNATQVVTDIGITDATVT ELIPGSSYTV EIFAQVGDGI KSLEPGRKSFCTDPASMASF DCEVVPKEPA LVLKWTCPPG ANAGFELEVSSGAWNNATHL ESCSSENGTE YRTEVTYLNF STSYNISITTVSCGKMAAPT RNTCTTGITD PPPPDGSPNI TSVSHNSVKVKFSGFEASHG PIKAYAVILT TGEAGHPSAD VLKYTYEDFKKGASDTYVTY LIRTEEKGRS QSLSEVLKYE IDVGNESTTLGYYNGKLEPL GSYRACVAGF TNITFHPQNK GLIDGAESYV SFSRYSDAVS LPQDPGVICG

NTAL is an integral transmembrane membrane protein of the plasmamembrane. The amino acid sequence of NTAL (Accession code: Q9GZY6; alsoknown as LAT2) is referred to herein as SEQ ID No.3, as follows:

[SEQ ID No. 3] MSSGTELLWP GAALLVLLGV AASLCVRCSR PGAKRSEKIYQQRSLREDQQ SFTGSRTYSL VGQAWPGPLA DMAPTRKDKLLQFYPSLEDP ASSRYQNFSK GSRHGSEEAY IDPIAMEYYNWGRFSKPPED DDANSYENVL ICKQKTTETG AQQEGIGGLCRGDLSLSLAL KTGPTSGLCP SASPEEDEES EDYQNSASIHQWRESRKVMG QLQREASPGP VGSPDEEDGE PDYVNGEVAA TEA

Preferably, the extracellular domain of NTAL is used as a biomarker ofsenescent cells. The amino acid sequence of the extracellular domain ofNTAL is referred to herein as SEQ ID No.4, as follows:

[SEQ ID No. 4] MSSGTE

EBP50 is an intracellular protein associated with the cytoplasmic faceof the plasma membrane. The amino acid sequence of EBP50 (Accessioncode: 014745; also known as NHERF1) is referred to herein as SEQ IDNo.5, as follows:

[SEQ ID No. 5] MSADAAAGAP LPRLCCLEKG PNGYGFHLHG EKGKLGQYIRLVEPGSPAEK AGLLAGDRLV EVNGENVEKE THQQVVSRIRAALNAVRLLV VDPETDEQLQ KLGVQVREEL LRAQEAPGQAEPPAAAEVQG AGNENEPREA DKSHPEQREL RPRLCTMKKGPSGYGFNLHS DKSKPGQFIR SVDPDSPAEA SGLRAQDRIVEVNGVCMEGK QHGDVVSAIR AGGDETKLLV VDRETDEFFKKCRVIPSQEH LNGPLPVPFT NGEIQKENSR EALAEAALESPRPALVRSAS SDTSEELNSQ DSPPKQDSTA PSSTSSSDPILDFNISLAMA KERAHQKRSS KRAPQMDWSK KNELFSNL

STX4 is a plasma membrane anchored protein with a large intracellulardomain and no extracellular domain. The amino acid sequence of STX4(Accession code: Q12846) is referred to herein as SEQ ID No.6, asfollows:

[SEQ ID No. 6] MRDRTHELRQ GDDSSDEEDK ERVALVVHPG TARLGSPDEEFFHKVRTIRQ TIVKLGNKVQ ELEKQQVTIL ATPLPEESMKQELQNLRDEI KQLGREIRLQ LKAIEPQKEE ADENYNSVNTRMRKTQHGVL SQQFVELINK CNSMQSEYRE KNVERIRRQLKITNAGMVSD EELEQMLDSG QSEVFVSNIL KDTQVTRQALNEISARHSEI QQLERSIREL HDIFTFLATE VEMQGEMINRIEKNILSSAD YVERGQEHVK TALENQKKAR KKKVLIAICV SITVVLLAVI IGVTVVG

VAMP3 is a plasma membrane anchored protein with a large intracellulardomain and no extracellular domain. The amino acid sequence of VAMP3(Accession code: Q15836; also known as Cellubrevin) is referred toherein as SEQ ID No.7, as follows:

[SEQ ID No. 7] MSTGPTAATG SNRRLQQTQN QVDEVVDIMR VNVDKVLERDQKLSELDDRA DALQAGASQF ETSAAKLKRK YWWKNCKMWA IGITVLVIFI IIIIVWVVSS

ARMCX-3 is an integral plasma membrane protein with two transmembranehelices and two extracellular domains. The amino acid sequence ofARMCX-3 (Accession code: Q9UH62; also known as ALEX3) is referred toherein as SEQ ID No.8, as follows:

[SEQ ID No. 8] MGYARKVGWV TAGLVIGAGA CYCIYRLTRG RKQNKEKMAEGGSGDVDDAG DCSGARYNDW SDDDDDSNES KSIVWYPPWARIGTEAGTRA RARARARATR ARRAVQKRAS PNSDDTVLSPQELQKVLCLV EMSEKPYILE AALIALGNNA AYAFNRDIIRDLGGLPIVAK ILNTRDPIVK EKALIVLNNL SVNAENQRRLKVYMNQVCDD TITSRLNSSV QLAGLRLLTN MTVTNEYQHMLANSISDFFR LFSAGNEETK LQVLKLLLNL AENPAMTRELLRAQVPSSLG SLFNKKENKE VILKLLVIFE NINDNFKWEENEPTQNQFGE GSLFFFLKEF QVCADKVLGI ESHHDFLVKV KVGKFMAKLA EHMFPKSQE

Preferably, both extracellular domains of ARMCX-3 are used as abiomarker of senescent cells. The amino acid sequence of the firstextracellular domain of ARMCX-3 is referred to herein as SEQ ID No. 9,as follows:

[SEQ ID No. 9] MGYARK

The amino acid sequence of the second extracellular domain of ARMCX-3 isreferred to herein as SEQ ID No. 10, as follows:

[SEQ ID No. 10] NRDIIRDLGGLPIVAKILNTRDPIVKEKALIVLNNLSVNAENQRRLKVYMNQVCDDTITSRLNSSVQLAGLRLLTNMTVTNEYQHMLANSISDFFRLFSAGNEETKLQVLKLLLNLAENPAMTRELLRAQVPSSLGSLFNKKENKEVILKLLVIFENINDNFKWEENEPTQNQFGEGSLFFFLKEFQVCADKVLGIESHHDFLVKVKVGKFMAKLAEHMFPKSQE

LANCL1 is an integral plasma membrane protein with six transmembranehelices and three extracellular domains. The amino acid sequence ofLANCL1 (Accession code: O43813) is referred to herein as SEQ ID No.11,as follows:

[SEQ ID No. 11] MAQRAFPNPY ADYNKSLAEG YFDAAGRLTP EFSQRLTNKIRELLQQMERG LKSADPRDGT GYTGWAGIAV LYLHLYDVFGDPAYLQLAHG YVKQSLNCLT KRSITFLCGD AGPLAVAAVLYHKMNNEKQA EDCITRLIHL NKIDPHAPNE MLYGRIGYIYALLFVNKNFG VEKIPQSHIQ QICETILTSG ENLARKRNFTAKSPLMYEWY QEYYVGAAHG LAGIYYYLMQ PSLQVSQGKLHSLVKPSVDY VCQLKFPSGN YPPCIGDNRD LLVHWCHGAPGVIYMLIQAY KVFREEKYLC DAYQCADVIW QYGLLKKGYGLCHGSAGNAY AFLTLYNLTQ DMKYLYRACK FAEWCLEYGEHGCRTPDTPF SLFEGMAGTI YFLADLLVPT KARFPAFEL

Preferably, all three extracellular domains of LANCL1 may be used as abiomarker of senescent cells. The amino acid sequence of the firstextracellular domain of LANCL1 is referred to herein as SEQ ID No.12, asfollows:

[SEQ ID No. 12] DVFGDPAYLQLAHGYVKQSLNCLTKR

The amino acid sequence of the second extracellular domain of LANCL1 isreferred to herein as SEQ ID No.13, as follows:

[SEQ ID No. 13] EKIPQSHIQQICETILTSGENLARKRNFTAKSPLMYEWYQEYYVGAAHGLAGIYYYLMQPSLQVSQGKLHSLVKPSVDYVCQLKFPSGNYPPCIGDNRD

The amino acid sequence of the third extracellular domain of LANCL1 isreferred to herein as SEQ ID No.14, as follows:

[SEQ ID No. 14] DMKYLYRACKFAEWCLEYGEHGCRTPDTP

B2MG is a secreted protein associated with the extra-cytoplasmic surfaceof the plasma membrane. The amino acid sequence of B2MG (Accession code:P61769) is referred to herein as SEQ ID No.15, as follows:

[SEQ ID No. 15] MSRSVALAVL ALLSLSGLEA IQRTPKIQVY SRHPAENGKSNFLNCYVSGF HPSDIEVDLL KNGERIEKVE HSDLSFSKDWSFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM

The first 20 amino acids of SEQ ID No.15 are the signal peptide of B2MG.The signal peptide of B2MG is responsible for directing B2MG to theplasma membrane of the cell for translocation across the plasma membraneto become a secreted protein. The amino acid sequence of B2MG withoutsignal peptide is referred to herein as SEQ ID No.16, as follows:

[SEQ ID No. 16] IQRTPKIQVY SRHPAENGKS NFLNCYVSGF HPSDIEVDLLKNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC RVNHVTLSQP KIVKWDRDM

PLD3 is a transmembrane protein with a single extracellular domain. Theamino acid sequence of PLD3 (Accession code: Q8IV08) is referred toherein as SEQ ID No.17, as follows:

[SEQ ID No. 17] MKPKLMYQEL KVPAEEPANE LPMNEIEAWK AAEKKARWVLLVLILAVVGF GALMTQLFLW EYGDLHLFGP NQRPAPCYDPCEAVLVESIP EGLDFPNAST GNPSTSQAWL GLLAGAHSSLDIASFYWTLT NNDTHTQEPS AQQGEEVLRQ LQTLAPKGVNVRIAVSKPSG PQPQADLQAL LQSGAQVRMV DMQKLTHGVLHTKFWVVDQT HFYLGSANMD WRSLTQVKEL GVVMYNCSCLARDLTKIFEA YWFLGQAGSS IPSTWPRFYD TRYNQETPMEICLNGTPALA YLASAPPPLC PSGRTPDLKA LLNVVDNARSFIYVAVMNYL PTLEFSHPHR FWPAIDDGLR RATYERGVKVRLLISCWGHS EPSMRAFLLS LAALRDNHTH SDIQVKLFVVPADEAQARIP YARVNHNKYM VTERATYIGT SNWSGNYFTETAGTSLLVTQ NGRGGLRSQL EAIFLRDWDS PYSHDLDTSA DSVGNACRLL

Preferably, the extracellular domain of PLD3 is used as a biomarker ofsenescent cells. The amino acid sequence of the extracellular domain ofPLD3 is referred to herein as SEQ ID No. 18, as follows:

[SEQ ID No. 18] QLFLWEYGDLHLFGPNQRPAPCYDPCEAVLVESIPEGLDFPNASTGNPSTSQAWLGLLAGAHSSLDIASFYWTLTNNDTHTQEPSAQQGEEVLRQLQTLAPKGVNVRIAVSKPSGPQPQADLQALLQSGAQVRMVDMQKLTHGVLsHTKFWVVDQTHEYLGSANMDWRSLTQVKELGVVMYNCSCLARDLTKIFEAYWFLGQAGSSIPSTWPRFYDTRYNQETPMEICLNGTPALAYLASAPPPLCPSGRTPDLKALLNVVDNARSFIYVAVMNYLPTLEFSHPHRFWPAIDDGLRRATYERGVKVRLLISCWGHSEPSMRAFLLSLAALRDNHTHSDIQVKLFVVPADEAQARIPYARVNHNKYMVTERATYIGTSNWSGNYFTETAGTSLLVTQNGRGGLRSQLEAIFLRDWDSPYSHDLDTSADSVGNACRLL

VPS26A is an intracellular protein associated with the cytoplasmic faceof the plasma membrane. The amino acid sequence of VPS26A (Accessioncode: O75436) is referred to herein as SEQ ID No.19, as follows:

[SEQ ID No. 19] MSFLGGFFGP ICEIDIVLND GETRKMAEMK TEDGKVEKHYLFYDGESVSG KVNLAFKQPG KRLEHQGIRI EFVGQIELFNDKSNTHEFVN LVKELALPGE LTQSRSYDFE FMQVEKPYESYIGANVRLRY FLKVTIVRRL TDLVKEYDLI VHQLATYPDVNNSIKMEVGI EDCLHIEFEY NKSKYHLKDV IVGKIYFLLVRIKIQHMELQ LIKKEITGIG PSTTTETETI AKYEIMDGAPVKGESIPIRL FLAGYDPTPT MRDVNKKFSV RYFLNLVLVDEEDRRYFKQQ EIILWRKAPE KLRKQRTNFH QRFESPESQA SAEQPEM

Thus, in one embodiment, preferably at least one polypeptide sequencecomprising an amino acid sequence substantially as set out in any one ofSEQ ID Nos. 1 to 19, or a variant or fragment thereof, is used as asenescent cell biomarker.

The inventors have found that the proteins DEP-1, NTAL, EBP50, STX4,VAMP-3, PLD3 and ARMCX-3 were specifically expressed in all senescentcells, whereas B2MG, LANCL1 and VPS26A were up-regulated only inp16-induced senescence. Therefore, preferably one or more of DEP-1,NTAL, EBP50, STX4, VAMP-3, PLD3 and ARMCX-3, or a variant or fragmentthereof, is used as a senescent cell biomarker. Hence, the at least onepolypeptide sequence comprises an amino acid sequence substantially asset out in any one of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 17 and18, or a variant or fragment thereof.

It is preferred however that the extracellular domains of any of theproteins described herein is used as a senescent cell biomarker. Theterm “fragment thereof” can therefore refer to an extracellular domainof the protein. Accordingly, preferably at least one polypeptidesequence comprising an amino acid sequence substantially as set out inany one of SEQ ID Nos. 2, 4, 9, 10, 12, 13, 14, 16 and 18, or a variantor fragment thereof, is used as an extracellular biomarker of senescentcells. Accordingly, one or more of DEP-1, NTAL, B2MG, ARMCX-3, PLD3 andLANCL1, or a variant or fragment thereof, is used as an extracellularbiomarker.

Most preferably, one or more of DEP-1, NTAL, ARMCX-3 and PLD3, or avariant or fragment thereof, is used as an extracellular biomarker.Hence, preferably the at least one polypeptide sequence comprises anamino acid sequence substantially as set out in any one of SEQ ID Nos.2, 4, 9, 10 and 18, or a variant or fragment thereof.

As described in the Examples, the inventors have demonstrated that anyof the ten senescent cell biomarkers described herein can be used tospecifically detect senescent cells in a biological sample.

Therefore, according to a second aspect, there is provided a method ofdetecting a senescent cell in a sample, the method comprises detectingthe expression, in the sample, of at least one senescent cell biomarkerselected from DEP-1, NTAL, EBP50, STX4, VAMP3, ARMCX-3, LANCL1, B2MG,PLD3 and VPS26A, or a variant or fragment thereof, wherein an increasedlevel of expression of the at least one biomarker or a variant orfragment thereof relative to the level of expression detected in areference sample is an indication of a senescent cell present in thesample.

The more biomarkers (or variants or fragments thereof) that are detectedin the sample, the greater the accuracy and reliability with whichsenescent cells can be identified. The method may therefore comprisedetecting two or more biomarkers, or variants or fragments thereof, inthe sample. In another embodiment, the method may comprise detectingthree or more biomarkers, or variants or fragments thereof, in thesample. Preferably, the method comprises detecting four, five, six ormore of the biomarkers, or variants or fragments thereof, describedherein. Most preferably, the method comprises detecting the presence ofDEP-1, NTAL, EBP50, STX4, VAMP-3, PLD3 and/or ARMCX-3, or a variant orfragment thereof. The method may comprise detecting one or more of thebiomarkers, according to the invention, in a sample together with otherknown biomarkers, such as DCR-2, Notch-3 or ICAM-1.

The term “detecting” can refer, but is not limited, to the use of anyone of the following conventional assays for detecting the presence ofone or more of the biomarkers, or variants or fragments thereof, in asample: flow cytometry; immunoassays, such as enzyme-linkedimmunosorbent assays (ELISAs), an enzyme immunoassay (EIAs),radioimmunoassay (RIAs), Western Blots, immuo-precipitation orimmunohistochemistry; chromogenic (enzyme activity) assays; fluorometricimaging plate reader (FLIPR) assay; or high performance liquidchromatography (HPLC) tandem mass spectrometry (MS/MS).

Preferably, the senescence biomarker is detected using flow cytometry.Advantageously, flow cytometry can be used to measure and distinguishbetween cell surface and intracellular localisation of a biomarkerprotein in situ. Intracellular localisation of biomarkers, according tothe invention, can be detected using flow cytometry by exposing thecells in the sample to a permeabilization agent, such as saponin, whichpermits entry of the cytometric antibodies into the target cells.

Alternatively, the presence of one or more of the senescence biomarkersmay be detected in the sample by measuring their functional activity,e.g. by enzyme assay. Alternatively, to measure the level of geneexpression of the senescence biomarkers, cDNA may be generated from mRNAextracted from cells present in the sample, and primers designed toamplify test sequences using a quantitative form of Polymerase ChainReaction.

The “sample” is preferably a bodily sample taken from a test subject.Detection for the presence of at least one senescent cell biomarker, ora variant or fragment thereof, in the sample, is therefore preferablycarried out in vitro. The sample may comprise blood, plasma, serum,spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostatefluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes,amniotic fluid, intraocular fluid, mucous, moisture in breath, animaltissue, cell lysates, tumour tissue, hair, skin, buccal scrapings,nails, bone marrow, cartilage, prions, bone powder, ear wax, orcombinations thereof.

In another embodiment, the sample may be contained within the testsubject, which may be an experimental animal (e.g. a mouse or rat) or ahuman, wherein the method is an in vivo based test. Alternatively, thesample may be an ex vivo sample or an in vitro sample. Therefore, thecells being tested may be in a tissue sample (for ex vivo based tests)or the cells may be grown in culture (an in vitro sample). Preferably,the biological sample is an ex vivo sample.

The method may comprise detecting the expression (or presence), in thesample, of the at least one senescent cell biomarker, wherein anincreased level of expression of the at least one biomarker or a variantor fragment thereof, relative to the level of expression detected in thereference sample is an indication of a senescent cell present in thesample. Preferably, the reference sample (i.e. a control) does notcomprise any senescent cells, and so does not express any of thebiomarkers, or only very low or undetectable concentrations thereof.

Expression of at least one of the senescence biomarkers or a variant orfragment thereof may be detected in any compartment of the cell (e.g. inthe nucleus, cytosol, the Endoplasmic Reticulum, the Golgi apparatus orthe intracellular surface of the plasma membrane), or on the cellsurface. Preferably, the senescence biomarker is expressed on the cellsurface or physically associated with the intracellular or extracellularsurface of the plasma membrane.

The inventors have developed a kit which is useful for detectingsenescent cells.

According to a third aspect, there is provided a senescent celldetection kit for detecting senescent cells in a sample, the kitcomprising means for detecting the presence, in a sample from a testsubject, of at least one senescent cell biomarker selected from DEP-1,NTAL, EBP50, STX4, VAMP3, ARMCX-3, LANCL1, B2MG, PLD3 and VPS26A, or avariant or fragment thereof.

It will be appreciated that the inventors have determined that there areten biomarkers which are associated with senescence, and the kits of theinvention may comprise means for detecting one or more of the senescencebiomarkers, or a variant or fragment thereof. The kit may thereforecomprise means for detecting: DEP-1, NTAL, EBP50, STX4, VAMP3, ARMCX-3,LANCL1, B2MG, PLD3 and VPS26A, or a variant or fragment thereof, orcombinations thereof.

Preferably, the kit comprises at least one control or reference sample.The kit may comprise a negative control and/or a positive control. Anegative control may comprise any non-senescent cell that does notexpress any of the senescent biomarkers according to the invention, oronly very low or undetectable concentrations thereof. A positive controlmay comprise any senescent cell that does express one or more of thesenescent biomarkers according to the invention.

Senescent biomarkers according to the invention, which do not contain anextracellular domain may be detected using conventional techniques knownin the art that are capable of detecting intracellular expression of aprotein, such as Western Blots, immuno-precipitation or flow cytometry(with the aid of a permeabilisation agent, such as saponin).

The kit may comprise a means to compare the level of expression (orconcentration) of the senescent biomarkers in the negative controlsample to the level of expression of the equivalent biomarkers in abiological sample from an unknown subject, wherein an increased level ofexpression of one or more of the biomarkers relative to that detected inthe negative control is an indication of senescence in the sample.Hence, by way of example, the concentration of the biomarker or afragment or variant thereof in a sample with a senescent cell may be atleast 1-, 2-, 5- or 10-fold high than in the negative control.

The inventors believe that the various senescence cell biomarkersdescribed herein can be harnessed in a cell targeting strategy forspecifically targeting and then killing senescent cells.

As such, according to a fourth aspect, there is provided a senescentcell biospecific drug conjugate for killing a senescent cell, theconjugate comprising a senescent cell targeting agent configured, inuse, to specifically target and bind to at least one senescent cellbiomarker selected from DEP-1, NTAL, EBP50, STX4, VAMP3, ARMCX-3,LANCL1, B2MG, PLD3 and VPS26A, or a variant or fragment thereof, and acytotoxic agent, which kills the bound senescent cell.

The senescent cell targeting agent may be an antibody or an antigenbinding fragment thereof, or an aptamer. Antibodies and fragmentsthereof represent preferred agents for use according to the invention.Antibodies according to the invention may be produced as polyclonal seraby injecting antigen into animals thereby producing polyclonalantibodies. Preferred polyclonal antibodies may be raised by inoculatingan animal (e.g. a rabbit) with antigen using techniques known to theart. Preferably, however, the antibody is a monoclonal antibody.Antibodies according to the invention may also comprise plasticantibodies. The term “plastic antibody” can mean molecularly imprintedpolymer nanoparticles (MIPs) with affinity for a target peptide orprotein. When monomers are polymerised in the presence of the selectedmolecular target, collective weak interactions between the monomers andthe target during polymerization result in the formation of populationsof complementary binding sites in the resulting polymer. This molecularimprinting approach has been previously used to target biologicallyrelevant molecules, including peptides and proteins. Binding affinityand selectivity of MIPs can be comparable to those of natural antibodiesand they have previously been shown to be effective in vivo. Moreover,MIPs can be conjugated with the desired cytotoxic drugs and thisapproach has also been shown to be efficient in the delivery of suchtoxic payloads to cells.

Preferred antibodies and epitope binding fragments thereof may haveimmunospecificity for any of the senescence biomarkers according to theinvention. Antibodies according to the invention may therefore be raisedagainst any one or more of SEQ ID Nos. 1-19, or a fragment or variantthereof.

Preferably, SEQ ID Nos. 2, 4, 9, 10, 12, 13, 14, 16 or 18, or a fragmentor variant thereof may be used as an antigen to create antibodies thatspecifically bind to senescent cells that display or express anextracellular biomarker according to the invention.

Functionally equivalent derivatives of the antibodies of the inventionare also encompassed and may comprise at least 75% sequence identity,more preferably at least 90% sequence identity, and most preferably atleast 95% sequence identity. It will be appreciated that most sequencevariation may occur in the framework regions (FRs), whereas the sequenceof the complementarity determining regions (CDRs) of the antibodiesshould be most conserved. For introduction into humans, the antibody maybe humanised, by splicing V region sequences (e.g. from a monoclonalantibody generated in a non-human hybridoma) with a C region (andideally FRs from the V region) sequences from human antibodies. Theresulting ‘engineered’ antibodies are less immunogenic in humans thanthe non-human antibodies from which they were derived and so are bettersuited for clinical use.

Preferably, the FR region of the antibody is conjugated or fused withthe cytotoxic agent, which may comprise a radioisotope, a toxin or atoxic peptide. The isotope may be any one selected from ¹³¹I or ⁹⁰Y. Thetoxin may be doxorubicin, calicheamicin, auristatin, maytansinoid,duocarmycin, or camptothecin analogues. The toxic peptide may bePseudomonas exotoxin A, diphtheria toxin, ricin, gelonin, saporin orpokeweed an antiviral protein.

Antibodies according to the invention specifically kill senescent cellsdue to their ability to specifically bind to senescent cells via theirCDR region(s). Therefore, the drug conjugate of the fourth aspect, andpreferably antibodies according to this aspect, may be use to treat ordelay the onset of age-related diseases.

In another embodiment, the targeting agent of the drug conjugate may bea small molecule. The small molecule may be capable of specificallybinding to an epitope of a biomarker that is expressed or displayed onthe surface of senescent cells. In another embodiment, the smallmolecule may comprise a means for gaining entry into senescent cells andspecifically binding to an epitope of a biomarker that is expressedintracellularly. The small molecule may have a weight of less than 1000Da.

It will be appreciated that drug conjugates according to this aspect ofthe invention may be used to specifically target and kill senescentcells that express or display senescent cell biomarkers, according tothe invention, on the intracellular or extracellular surface of theirplasma membrane.

In embodiments where the drug conjugate is intended to target abiomarker, which is expressed or displayed on the extracellular surfaceof senescent cells, the targeting agent of the drug conjugate may be anantibody comprising CDRs that specifically binds to an extracellularepitope of the biomarker.

In embodiments where the drug conjugate is intended to target asenescent cell biomarker, which is only expressed intracellularly, thetargeting agent of the drug conjugate may be a small molecule that iscapable of gaining entry into senescent cells and specifically bindingto an epitope of the biomarker.

The senescent cell biomarkers according to the invention may be used toidentify senescent cells, which can be targeted, for example, by thedrug conjugate according to this aspect of the invention, for treatmentof conditions associated with cell senescence, such as ageing andcancer.

Hence, in a fifth aspect, there is provided the senescent cellbiospecific drug conjugate according to the fourth aspect, for use as amedicament.

In a sixth aspect, there is provided the senescent cell biospecific drugconjugate according to the fourth aspect, for use in treating,preventing or ameliorating an age-related disease.

In a seventh aspect, there is provided a method of treating, preventingor ameliorating an age-related disease, the method comprisingadministering, to a subject in need of such treatment, a therapeuticallyeffective amount of the senescent cell biospecific drug conjugateaccording to the fourth aspect.

Age-related diseases may include but are not limited to impaired woundhealing, dermal thinning, arterial wall stiffening, atherosclerosis,cardiovascular disease, cancer, arthritis, glaucoma, cataracts,osteoporosis, type 2 diabetes, hypertension, Alzheimer's disease andother types of dementia.

According to an eighth aspect, there is provided an age-related diseasetreatment pharmaceutical composition comprising the senescent cellbiospecific drug conjugate according to the fourth aspect and apharmaceutically acceptable vehicle.

According to a ninth aspect, there is provided a method of specificallykilling senescent cells, the method comprising:

-   -   (i) determining the presence of a senescence cell in a subject;        and    -   (ii) administering, to a subject, a therapeutically effective        amount of the senescent cell biospecific drug conjugate        according to the fourth aspect.

The method of the ninth aspect of the invention may be used tospecifically kill senescent cells in vivo, in vitro or ex vivo.

The methods, kits, conjugates and compositions according to theinvention preferably comprise the use of at least one of the polypeptidesequences substantially as set out in any one of SEQ ID Nos. 1 to 19, ora fragment or variant thereof, as a biomarker of a senescent cell.Preferably, at least one of the polypeptide sequences substantially asset out in any one of SEQ ID Nos. 2, 4, 9, 10, 12, 13, 14, 16 or 18, ora fragment or variant thereof is used as an extracellular biomarker ofsenescent cells.

It will be appreciated that agents, conjugates, antibodies andcompositions according to the invention may be used in a medicamentwhich may be used in a monotherapy, for treating or delaying the onsetof age-related diseases. Alternatively, such agents according to theinvention may be used as an adjunct to, or in combination with, knowntherapies for treating or delaying the onset of age-related diseases.

The agents and antibodies according to the invention may be combined incompositions having a number of different forms depending, inparticular, on the manner in which the composition is to be used. Thus,for example, the composition may be in the form of a powder, tablet,capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray,micellar solution, transdermal patch, liposome suspension or any othersuitable form that may be administered to a person or animal in need oftreatment. It will be appreciated that the vehicle of medicamentsaccording to the invention should be one which is well-tolerated by thesubject to whom it is given.

Medicaments comprising agents and antibodies according to the inventionmay be used in a number of ways. For instance, oral administration maybe required, in which case the agents may be contained within acomposition that may, for example, be ingested orally in the form of atablet, capsule or liquid. Compositions comprising agents of theinvention may be administered by inhalation (e.g. intranasally).Compositions may also be formulated for topical use. For instance,creams or ointments may be applied to the skin.

Agents, compositions and antibodies according to the invention may alsobe incorporated within a slow- or delayed-release device. Such devicesmay, for example, be inserted on or under the skin, and the medicamentmay be released over weeks or even months. The device may be located atleast adjacent to the treatment site. Such devices may be particularlyadvantageous when long-term treatment with agents used according to theinvention is required and which would normally require frequentadministration (e.g. at least daily injection).

In a preferred embodiment, agents, compositions and antibodies accordingto the invention may be administered to a subject by injection into theblood stream or directly into a site requiring treatment. For example,the medicament may be injected at least adjacent to a senescent cell, orwithin a tumour. Injections may be intravenous (bolus or infusion) orsubcutaneous (bolus or infusion), or intradermal (bolus or infusion).

It will be appreciated that the amount of the agent, composition andantibody that is required is determined by its biological activity andbioavailability, which in turn depends on the mode of administration,the physicochemical properties of the modulator and whether it is beingused as a monotherapy or in a combined therapy. The frequency ofadministration will also be influenced by the half-life of the agent orantibody within the subject being treated. Optimal dosages to beadministered may be determined by those skilled in the art, and willvary with the particular agent in use, the strength of thepharmaceutical composition, the mode of administration, and theadvancement of the senescence-associated disease(s). Additional factorsdepending on the particular subject being treated will result in a needto adjust dosages, including subject age, weight, gender, diet, and timeof administration.

Generally, a daily dose of between 0.01 μg/kg of body weight and 500mg/kg of body weight of the agent according to the invention may be usedfor treating, ameliorating, or preventing senescence-associated disease,depending upon which agent is used. More preferably, the daily dose isbetween 0.01 mg/kg of body weight and 400 mg/kg of body weight, morepreferably between 0.1 mg/kg and 200 mg/kg body weight, and mostpreferably between approximately 1 mg/kg and 100 mg/kg body weight.

The agent, composition or antibody may be administered before, during orafter onset of the senescence-associated disease. Daily doses may begiven as a single administration (e.g. a single daily injection).Alternatively, the agent may require administration twice or more timesduring a day. As an example, agents may be administered as two (or moredepending upon the severity of the disease being treated) daily doses ofbetween 25 mg and 7000 mg (i.e. assuming a body weight of 70 kg). Asubject receiving treatment may take a first dose upon waking and then asecond dose in the evening (if on a two dose regime) or at 3- or4-hourly intervals thereafter. Alternatively, a slow release device maybe used to provide optimal doses of agents according to the invention toa patient without the need to administer repeated doses.

Known procedures, such as those conventionally employed by thepharmaceutical industry (e.g. in vivo experimentation, clinical trials,etc.), may be used to form specific formulations comprising the agentsaccording to the invention and precise therapeutic regimes (such asdaily doses of the agents and the frequency of administration).

A “therapeutically effective amount” of agent is any amount which, whenadministered to a subject, is the amount of the agent, the compositionor antibody that is needed to treat the senescence-associated disease,or produce the desired effect, such as inhibiting senescence cellformation.

For example, the therapeutically effective amount of agent used may befrom about 0.01 mg to about 800 mg, and preferably from about 0.01 mg toabout 500 mg. It is preferred that the amount of agent is an amount fromabout 0.1 mg to about 250 mg, and most preferably from about 0.1 mg toabout 20 mg.

A “pharmaceutically acceptable vehicle” as referred to herein, is anyknown compound or combination of known compounds that are known to thoseskilled in the art to be useful in formulating pharmaceuticalcompositions.

In one embodiment, the pharmaceutically acceptable vehicle may be asolid, and the composition may be in the form of a powder or tablet. Asolid pharmaceutically acceptable vehicle may include one or moresubstances which may also act as flavouring agents, lubricants,solubilisers, suspending agents, dyes, fillers, glidants, compressionaids, inert binders, sweeteners, preservatives, dyes, coatings, ortablet-disintegrating agents. The vehicle may also be an encapsulatingmaterial. In powders, the vehicle is a finely divided solid that is inadmixture with the finely divided active agents according to theinvention. In tablets, the active agent (e.g. the peptide or antibody)may be mixed with a vehicle having the necessary compression propertiesin suitable proportions and compacted in the shape and size desired. Thepowders and tablets preferably contain up to 99% of the active agents.Suitable solid vehicles include, for example calcium phosphate,magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin,cellulose, polyvinylpyrrolidone, low melting waxes and ion exchangeresins. In another embodiment, the pharmaceutical vehicle may be a geland the composition may be in the form of a cream or the like.

However, the pharmaceutical vehicle may be a liquid, and thepharmaceutical composition is in the form of a solution. Liquid vehiclesare used in preparing solutions, suspensions, emulsions, syrups, elixirsand pressurized compositions. The active agent according to theinvention may be dissolved or suspended in a pharmaceutically acceptableliquid vehicle such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid vehicle can containother suitable pharmaceutical additives such as solubilisers,emulsifiers, buffers, preservatives, sweeteners, flavouring agents,suspending agents, thickening agents, colours, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid vehicles fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the vehicle can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid vehicles are useful insterile liquid form compositions for parenteral administration. Theliquid vehicle for pressurized compositions can be a halogenatedhydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions, which are sterile solutions orsuspensions, can be utilised by, for example, intramuscular,intrathecal, epidural, intraperitoneal, intravenous and particularlysubcutaneous injection. The agent, composition or antibody may beprepared as a sterile solid composition that may be dissolved orsuspended at the time of administration using sterile water, saline, orother appropriate sterile injectable medium.

The agents and compositions of the invention may be administered orallyin the form of a sterile solution or suspension containing other solutesor suspending agents (for example, enough saline or glucose to make thesolution isotonic), bile salts, acacia, gelatin, sorbitan monoleate,polysorbate 80 (oleate esters of sorbitol and its anhydridescopolymerized with ethylene oxide) and the like. The agent, antibody orcomposition according to the invention can also be administered orallyeither in liquid or solid composition form. Compositions suitable fororal administration include solid forms, such as pills, capsules,granules, tablets, and powders, and liquid forms, such as solutions,syrups, elixirs, and suspensions. Forms useful for parenteraladministration include sterile solutions, emulsions, and suspensions.

It will be appreciated that the invention extends to any nucleic acid orpeptide or variant, derivative or analogue thereof, which comprisessubstantially the amino acid or nucleic acid sequences of any of thesequences referred to herein, including variants or fragments thereof.The terms “substantially the amino acid/nucleotide/peptide sequence”,“variant” and “fragment”, can be a sequence that has at least 40%sequence identity with the amino acid/nucleotide/peptide sequences ofany one of the sequences referred to herein, for example 40% identitywith the polypeptide identified as SEQ ID Nos. 1-19, and so on.

Amino acid/polynucleotide/polypeptide sequences with a sequence identitywhich is greater than 50%, more preferably greater than 65%, 70%, 75%,and still more preferably greater than 80% sequence identity to any ofthe sequences referred to are also envisaged. Preferably, the aminoacid/polynucleotide/polypeptide sequence has at least 85% identity withany of the sequences referred to, more preferably at least 90%, 92%,95%, 97%, 98%, and most preferably at least 99% identity with any of thesequences referred to herein.

The skilled technician will appreciate how to calculate the percentageidentity between two amino acid/polynucleotide/polypeptide sequences. Inorder to calculate the percentage identity between two aminoacid/polynucleotide/polypeptide sequences, an alignment of the twosequences must first be prepared, followed by calculation of thesequence identity value. The percentage identity for two sequences maytake different values depending on:—(i) the method used to align thesequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman(implemented in different programs), or structural alignment from 3Dcomparison; and (ii) the parameters used by the alignment method, forexample, local vs global alignment, the pair-score matrix used (e.g.BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional formand constants.

Having made the alignment, there are many different ways of calculatingpercentage identity between the two sequences. For example, one maydivide the number of identities by: (i) the length of shortest sequence;(ii) the length of alignment; (iii) the mean length of sequence; (iv)the number of non-gap positions; or (iv) the number of equivalencedpositions excluding overhangs. Furthermore, it will be appreciated thatpercentage identity is also strongly length dependent. Therefore, theshorter a pair of sequences is, the higher the sequence identity one mayexpect to occur by chance.

Hence, it will be appreciated that the accurate alignment of protein orDNA sequences is a complex process. The popular multiple alignmentprogram ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22,4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882)is a preferred way for generating multiple alignments of proteins or DNAin accordance with the invention. Suitable parameters for ClustalW maybe as follows: For DNA alignments: Gap Open Penalty=15.0, Gap ExtensionPenalty=6.66, and Matrix=Identity. For protein alignments: Gap OpenPenalty=10.0, Gap Extension Penalty=0.2, and Matrix=Gonnet. For DNA andProtein alignments: ENDGAP=−1, and GAPDIST=4. Those skilled in the artwill be aware that it may be necessary to vary these and otherparameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two aminoacid/polynucleotide/polypeptide sequences may then be calculated fromsuch an alignment as (N/T)*100, where N is the number of positions atwhich the sequences share an identical residue, and T is the totalnumber of positions compared including gaps but excluding overhangs.Hence, a most preferred method for calculating percentage identitybetween two sequences comprises (i) preparing a sequence alignment usingthe ClustalW program using a suitable set of parameters, for example, asset out above; and (ii) inserting the values of N and T into thefollowing formula:—Sequence Identity=(N/T)*100.

Alternative methods for identifying similar sequences will be known tothose skilled in the art. For example, a substantially similarnucleotide sequence will be encoded by a sequence which hybridizes toany sequences referred to herein or their complements under stringentconditions. By stringent conditions, we mean the nucleotide hybridisesto filter-bound DNA or RNA in 3× sodium chloride/sodium citrate (SSC) atapproximately 45° C. followed by at least one wash in 0.2×SSC/0.1% SDSat approximately 20-65° C. Alternatively, a substantially similarpolypeptide may differ by at least 1, but less than 5, 10, 20, 50 or booamino acids from the sequences shown in SEQ ID Nos.1-19.

Due to the degeneracy of the genetic code, it is clear that any nucleicacid sequence described herein could be varied or changed withoutsubstantially affecting the sequence of the protein encoded thereby, toprovide a variant thereof. Suitable nucleotide variants are those havinga sequence altered by the substitution of different codons that encodethe same amino acid within the sequence, thus producing a silent change.Other suitable variants are those having homologous nucleotide sequencesbut comprising all, or portions of, sequence, which are altered by thesubstitution of different codons that encode an amino acid with a sidechain of similar biophysical properties to the amino acid itsubstitutes, to produce a conservative change. For example smallnon-polar, hydrophobic amino acids include glycine, alanine, leucine,isoleucine, valine, proline, and methionine. Large non-polar,hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.The polar neutral amino acids include serine, threonine, cysteine,asparagine and glutamine. The positively charged (basic) amino acidsinclude lysine, arginine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. It will thereforebe appreciated which amino acids may be replaced with an amino acidhaving similar biophysical properties, and the skilled technician willknow the nucleotide sequences encoding these amino acids.

All of the features described herein (including any accompanying claims,abstract and drawings), and/or all of the steps of any method or processso disclosed, may be combined with any of the above aspects in anycombination, except combinations where at least some of such featuresand/or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodimentsof the same may be carried into effect, reference will now be made, byway of example, to the accompanying Figures, in which:—

FIG. 1 shows the analysis of the membrane fraction of EJp16 and EJp21cells without and with induction of p16 and p21 respectively. A) Westernblots of EJp16 and EJp21 in the presence or absence of tet(tetracycline), showing the induction of expression of exogenous p16 orp21, respectively. B) SA-β-Gal staining of EJp16 and EJp21 cellsuninduced (Control) or 4 days after tet removal to induce expression ofexogenous p16 or p21 (Senescent). Blue staining and morphologicalchanges are indicative of senescence. C) Western blot analysis oflysates separated into cytosolic and membrane fractions of EJp21 andEJp16 cells uninduced (C) or 4 days after tet removal (S). Calnexin isused as a marker of membrane proteins and MAPK as a marker of thecytosolic fraction;

FIG. 2 shows proteomic screening of membrane proteins in senescentcells. A) Graphic representation of mass spec hits in EJp21 and EJp16control and senescent cells. B) Number of membrane proteinsdifferentially expressed in control and senescent EJp21 and EJp16,compared to those present in both conditions.

FIG. 3 is a Western blot validation of senescent-specific targets. A)and B) show protein expression of selected targets in the membranefraction of lysates from EJp16 and EJp21 uninduced (C) or 4 days aftertet removal (S). Calnexin and Na/K ATPase are used as loading controls;

FIG. 4 shows the expression of selected targets in membranes ofsenescent cells. Sucrose gradient fractionation of the membrane fractionof lysates from EJp164 days after tet removal. Calnexin and Na/K ATPaseare used as markers of the cell membrane fractions. HDAC1 is used asmarker of the nuclear fraction. MAPK is used as marker of the cytosolicfractions. SOD is used as marker of the mitochondrial fraction;

FIG. 5 shows the expression and localization of the novel senescencemarkers. Immunofluorescent images of selected targets in EJp16 and EJp21uninduced (Control) or4 days after tet removal (Senescent), as well asearly passage normal human diploid fibroblasts compared to thoseentering replicative senescence;

FIG. 6 relates to defining a new protocol for the detection of senescentcells. A) Representative plot analysis of fluorescence levels in controland senescent EJp16, HT1080p21-9 and human diploid fibroblasts (HDF)stained with fluorescently tagged antibodies against B2MG, DEP-1 andNOTCH3, as measured by flow cytometry. Senescent cells were analysedafter 5 days of p16 or p21 expression. B) Average fold increases of meanfluorescence intensity (MFI) of the same cells. Experiments wereperformed in triplicate. Error bars show standard deviation; and

FIG. 7 is SA-β-Gal staining of control and senescent IMR90, HT1080p21-9(after 4 days of p21 expression following exposure to IPTG) and normalhuman diploid fibroblasts (HDF).

EXAMPLES

The inventors have studied the expression profile of plasma membraneproteins in senescent cells in order to identify novel markers thatcould be easily recognized and propose potential effectors andmodulators of the senescent pathway. Ten novel specific markers ofsenescence were validated (Examples 1 and 2), and two of these wereselected in order to develop a fast and straightforward FACS-basedapproach to identify senescent cells (Example 3).

Materials and Methods

Cell Culture

The EJ human bladder cancer cell lines were maintained in DMEMsupplemented with 10% fetal bovin serum (FBS) (Gibco), and pen-strep (50unit/ml). EJ p21 and EJp53 cells were maintained with hygromycin (100μg/ml) and genticin (750 μg/ml) plus (1 μg/ml) tetracycline. EJp16 cellswere maintained with hygromycin (100 μg/ml) and puromycin (2 μg/ml) plus(1 μg/ml) tetracycline. In order to inhibit p21 and p16 expression,tetracycline (tet) was added to the medium every 3 days to finalconcentration (1 μg/ml). To induce p21, p16 and p53 expression, cellswere washed three times and seeded directly in culture medium in theabsence of tet (Fang et al., 1999). IMR90 (human fibroblasts wad derivedfrom lungs of a 16-weeks female fetus) and 501T (human fibroblast whichis driven from normal human skin) these fibroblasts were cultured untilthey reached the end of their replicative senescence. Restrictivedermopathy (RD) cells were kindly provided by Dr Sue Shackleton. Toinduce p21 expression in HT1080p21, 100 μM IPTG was added to the medium.

Plasma Membrane Protein Extraction

This method was performed according to the Abcam Plasma Membrane Proteinextraction Kit (ab65400).

SDS-PAGE Separation

Senescent and growing EJp21 and EJp16 plasma membrane samples wereseparated by 10% SDSPAGE. After staining with the Coomassie blue, thegel was cut to obtain separate sample lanes. Each gel strip was thensliced into 50 slices, from the loading well down to the bottom of thegel. The proteins in the gel bands were digested with trypsin accordingto the protocol described previously (Shevchenko et al, 2006).

Extraction and Analysis of Proteins from Gel Lanes by Mass Spectrometry(Synapt G2S). Gel lanes were cut sequentially into slices ofapproximately 1.5 mm and transferred to a 96 well low binding PCR plate.Each slice was washed/swollen with ammonium bicarbonate (80 ul, 50 mM)for 30 minutes, after this time the buffer was aspirated off using aGilson. Each slice was destained with acetonitrile (80 ul) for 30minutes, the solvent was removed. Steps 2) and 3) were repeated. Afteraspiration of the final acetonitrile, 15 ul of sequencing grade modifiedtrypsin V511 (Promega), 20 ug/1.8 ml 25 mM ammonium bicarbonate, wasadded to each dehydrated gel piece. The plate was sealed and heated at30° C. overnight. The sealing film was removed and extraction bufferadded to each well (80 μl, 97% TFA (0.2%) 3% acetonitrile). The sampleswere extracted at room temperature for 1 hour. The extracted sampleswere transferred to low-binding eppendorf tubes and concentrated todryness in a speedvac. The samples were redissolved in injection solvent(40 ul, 5% TFA) and analysed by mass spectrometry. Nanoscale LC was usedto separate the complex peptide mixtures using a Waters nanoACQUITYUPLC. Chromatography was performed using a 50 minute reversed phasegradient (formic acid (0.1%)/acetonitrile) and a 75 μm×25 cm C18 column(Waters, BE130) operated at 300 nL/min. Mass spectrometry analysis wasperformed using a SYNAPT G25 (Waters Manchester UK) operated in adata-independent (MSE) manner. The selected analysis mode enabledprecursor and fragment ions from the tryptic digest to be analysedsimultaneously. The data acquired was processed and searched usingProteinLynx Global Server (Waters) and visualized and reanalyzed usingScaffold (Proteome Software, Oregon, USA).

Senescence-associated-β-Galactosidase (SA-β-Gal) Staining

Cells were washed three times with PBS, and fixed with 4% formaldehydefor 5 min at room temperature. The detail of SA-β-gal staining wasdescribed previously (Dimri et al, 1995).

Immunoblot Analysis

Extracellular membrane samples were extracted and 1 μg/ml ProteaseInhibitor Cocktail Set III (Calbiochem) added to the samples. Proteinconcentrations were then determined using Bradford protein assay(Fermentas). 20 μg of total cell protein per sample were subjected to10% or 6% SDS-PAGE and transferred to Immobilon-P membrane (Millipore).An ECL detection system (Thermo Scientific) was used.

Immunofluorescence

Cells were split into 6-well plates containing sterile coverslips. After24 hours, media was aspirated from the plates and cells were washedthree times with 1×PBS. Cells were fixed using 1 ml of 4%paraformaldehyde for 30 min with gentle shaking. After fixing, cellswere washed three times with 1×PBS and permeabilised with 1 ml 0.1%Triton X-100 for 10 minutes. Cells were then washed three times with1×PBS and blocked with 1% BSA for 30 minutes. Coverslips were incubatedwith 100 μl 1:100 primary antibody overnight at 4° C. The following day,coverslips were washed three times with 1×PBS and incubated with 100 μlsecondary anti-rabbit and anti-mouse antibody (Alexa Fluor 488 and 594,Invitrogen) for 45 minutes in the dark. After incubation, coverslipswere washed three times with 1×PBS and stained with4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Invitrogen) for 10minutes. Slides were labelled and the coverslips were mounted and sealedwith transparent nail varnish. Slides were analysed using a Nokia TE300semi-automatic microscope.

FACS Analysis

Plates were washed with cold 1×PBS, and then the cells were collected bygently scraping them in 0.5 ml cold 1×PBS on ice. Trypsin should not beused because it leads to loss of extracellular proteins. The cells werethen spun down at 200 g for 5 min at 4° C. The supernatant wasdiscarded, the cells were re-suspend in 200 μl of blocking buffer (0.5%BSA+1×PBS) and then they were incubated on ice for 15 min. The cellswere transferred into a 96 rounded bottom multi-well plate and spun downat 500 g for 5 min at 4° C. Once again, the supernatant was discarded.The pellet was re-suspend in an Antibody Mix and incubated at 4° C. inthe dark for 30-45 min. The cells were then washed twice with BlockingBuffer (150 μl per well) followed by a spin at 500 g for 5 min at 4° C.The supernatant was discarded and the pellet was re-suspended in 300-500μl of Blocking Buffer. Cellular fluorescence was detected using acytometer.

Example 1—Proteomic Analysis of the Expression of Membrane Proteins inSenescent Cells

In order to characterize the profile of proteins selectively expressedin the plasma membrane of senescent cells, the inventors used a bladdercancer cell line, EJ, with a tet-regulatable p21 or p16 expressionsystem (see FIG. 1A). These cells, named EJp21 and EJp16, respectively(Fang et al, 1999; Macip et al, 2002), irreversibly senesce afterprolonged expression of the induced protein (see FIG. 1B). The membranefraction was isolated form lysates of these proteins (see FIG. 1C) and amass spectrometry screen performed to compare the senescent cells totheir non-senescent counterparts. As shown in FIG. 2, 107 proteins wereexclusively present in membranes of senescent EJp21 and 132 in EJp16.From these lists, ten proteins were selected for further validation:DEP-1, NTAL, EBP50, STX4, VAMP-3, ARMX-3, B2MG, LANCL1, VPS26A and PLD3.They were all chosen because they had not previously been shown to beassociated with senescence and are all plasma membrane-associatedproteins. None of the selected proteins had known functions that couldimmediately predict their mechanistic involvement in the senescentpathway. Of note, the screen also detected DCR-2, Notch-3 and ICAM1, allof which had been previously associated with senescence, which confirmsthe suitability of the screening protocols used.

Example 2—Validation of Potential Membrane Markers of Senescent Cells

The inventors next confirmed that the ten selected proteins (listed inExample 1) were indeed expressed preferentially in the membranes ofsenescent cells. To this end, the cell membrane fraction of lysates fromEJp16 and EJp21 that had been induced to senesce were used. As shown inFIG. 3A, basal levels of DEP-1, NTAL, EBP50, STX4, VAMP3 and ARMCX-3were low in uninduced EJp16 cells. After 5 days of p16 expression, whencells are known to be irreversibly senescent (Macip et al, 2002),expression of these proteins was highly increased, except for STX4 andVAMP-3, which only showed a minor induction. DEP-1 and NTAL were notablyexpressed in EJp21 in basal conditions, but were still up-regulatedafter the p21 induction for 5 days. NTAL, EBP50, STX4, VAMP-3 andARMCX-3 all had low basal levels and a substantial increase inexpression after EJp21 entered senescence. As shown in FIG. 3B, B2MG,LANCL1 and VPS26A underwent moderate increases in response to p16, butnot p21. Also, PLD3 did not show any expression change in any modeltested. Finally, DCR-2 was shown to be induced in both p16- andp21-dependent senescence, as expected. All of these results togetherconfirmed that five of the potential markers (DEP-1, NTAL, EBP50, STX4,VAMP-3 and ARMCX-3) were specifically expressed in senescent cells,although at different levels, and three more (B2MG, LANCL1 and VPS26A)were up-regulated only in p16-induced senescence.

The inventors further confirmed these results using cell fractionationof EJp16 cell lysates by sucrose gradient. FIG. 4 shows that DEP-1,NTAL, EBP50, STX4, ARMCX-3 and B2MG co-localize in the same fraction ascell membrane markers Na/K ATPase and Calnexin. This underscores thehypothesis that these proteins are present in membrane of senescentcells. Immunofluorescent microscopy was also used to study theexpression and localization of these proteins (see FIG. 5). DEP-1, NTAL,EBP50 and STX4 showed induction in senescent EJp16, as compared to thepositive control (DCR-2). VAMP-3 and ARMCX-3 also showed up-regulation,but at lower levels. In EJp21, all markers were significantly increased.The expression of these proteins in IMR90 human fibroblasts was alsomeasured, comparing early passage cells to those induced to senesceafter serial passaging (see FIG. 7). All the proteins tested showed lowbasal levels in growing fibroblasts and increased expression insenescent ones (see FIG. 5), confirming that they could be used asmarkers of replicative senescence in normal cells.

Example 3—Characterization of Senescence Markers by FACS Analysis

With the information from the validation experiments (i.e. Example 2),the inventors chose two of the novel membrane proteins (DEP-1 and B2MG)to define a simple and specific protocol, using flow cytometry, thatwould allow for the rapid detection of senescent cells in culture. DEP-1and B2MG were initially chosen because they had large extracellularepitopes recognized by commercially available fluorescent-taggedantibodies. NOTCH3 was used as a positive control. All three antibodieswere mixed and incubated with non-permeabilized cells (see Materials andMethods for protocol details). The total time needed to measure thepresence of senescent cells in cell cultures was under 2 hours. As shownin FIG. 6, there was a consistent 2- to 3-fold increase in all of themarkers in EJp16 after the induction of senescence. This result wasconfirmed using another model of p21-induced senescence HT1080p21-9(Chang et al, 2000; Masgras et al, 2012) (see FIG. 7), which showedapproximately a 3-fold increase in cell surface expression in each ofthe three markers. Moreover, normal human diploid fibroblasts thatentered replicative senescence also showed up-regulation of the markers,although at lower levels (FIG. 6), which is consistent with a lowerpercentage of SA-β-Gal positive cells (see FIG. 7). These resultsconfirm that the validated membrane markers of senescence from theinventors proteomic screen can be successfully used to determine thepresence of senescent cells in culture and could provide a faster andmore selective detection tool than those currently available.

Discussion

Senescence is a well-defined cellular mechanism with a critical role inprocesses as diverse as cancer and ageing. Despite having been studiedfor decades, the mechanisms involved in senescence are not fullyunderstood. One of the features of senescent cells that had not beenpreviously characterized was the profile of expression of proteins ontheir surface. Such proteins have the potential to be especiallyrelevant for three reasons. Firstly, these proteins could contribute toexplaining how these cells interact with the microenvironment and alsoaid our understanding of the mechanisms of senescent cell clearance.This is important in the context of the tumour suppressor functions ofsenescence as well as its involvement in the symptoms associated withageing (Baker et al, 2011). Secondly, specific cell membrane proteinswith extracellular epitopes would be useful for rapidly detectingsenescent cells in a laboratory environment. Given that the currentprotocols for these analyses are far from ideal, identifyingextracellular epitopes of the senescent proteome could greatly improvethis field of study. Finally, uncovering novel up-regulated proteinscould enhance our understanding of the processes that determine theestablishment and maintenance of the senescent phenotype.

Using a proteomics approach, the inventors identified and validated tenproteins expressed at higher levels in plasma membrane fractions ofsenescent cells than in controls. Six of the proteins have at least oneextracellular domain or are associated with the plasma membrane. Fromtheir known functions, it is not immediately clear what role they couldplay in senescence. DEP-1 participates in cell adhesion, which coulddetermine how senescent cells interact with their microenvironment. STX4and VAMP-3 contribute to vesicle traffic in cells, perhaps impinging onsome aspects of the SASP. NTAL, EBP50 and LANCL1 belong to differentsignalling pathways that could be linked to senescence. B2MG and VPS26Ahave roles in the immune system, and this could be related to theclearance of senescent cells from tissues. ARMCX-3 has a potentialtumour suppressor effect that could perhaps be explained by its role ininducing senescence. Finally, PLD3's phospholipase activity may beinvolved in senescence through unknown mechanisms. Further experimentsto determine whether any of these proteins actively contribute to thesenescent phenotype (or if their upregulation is just an epiphenomenon)are currently being performed.

All 10 targets were studied in different models, mainly the inducible EJcell lines that undergo senescence through activation of one of the mainpathways involved in the process, p16 or p21. All of them wereup-regulated in at least one of the models, with most clearly induced inboth. Moreover, the results were also validated in normal humanfibroblasts, thus confirming the relevance of the data in bothreplicative and stress-induced models of senescence.

The inventors have proven that these proteins, specifically the six thatshowed better induction (DEP-1, NTAL, EBP50, STX4, VAMP-3, ARMCX-3 andB2MG), have the potential to be used as surrogate markers of senescence,together with those previously described (p21, p16, p15, DCR2, NOTCH-3,etc.). As a proof of principle, they selected two of the six proteins,DEP-1 and B2MG, to develop a staining protocol that could help determinethe amount of senescent cells present in a sample. The goal was toachieve higher specificity and shorter experimental times than thecurrent gold standard, the SA-β-Gal assay. The inventors believe thattheir results show that such a detection method, based on specificantibodies against extracellular epitopes, is feasible and successful.Results can be obtained under 2 hours, compared with the overnightincubation times needed for the classic SA-β-galactosidase staining.Further optimization will be required to determine the best targets andconditions. Increasing the simultaneous number of markers detected couldaugment the specificity of the protocol, if needed. Also, markers morespecific to either the p16 or p21 pathways could help determine which ofthe two pathways is preferentially activated in response to eachsenescence-inducing stimulus.

This proteomic screen provides new information about the mechanismsinvolved in senescence and can be used experimentally to rapidly detectsenescent cells. Moreover, the inventors hope that further studies, inthe future, will determine the exact role of these novel markers in thesenescent pathways, thus contributing to our understanding of thisintricate cellular process. Such information could be important todefine new therapeutic interventions that could increase the positiveimpact of senescence on human health and/or diminish its negativeeffects.

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The invention claimed is:
 1. A method of targeting a senescent cell witha biospecific drug conjugate in a subject, the method comprisingadministering to the subject a therapeutically effective amount of asenescent cell biospecific drug conjugate, wherein the senescent cellbiospecific drug conjugate comprises i) an antibody or anantigen-binding fragment thereof that specifically targets and binds toDEP-1, and ii) a cytotoxic agent, which kills the bound senescent cells,wherein DEP-1 comprises an amino acid sequence that has at least 90%sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ IDNO: 2, and wherein the subject comprises a senescent cell.
 2. The methodaccording to claim 1, wherein the subject is suffering fromcardiovascular disease, cataracts, osteoporosis, type 2 diabetes,hypertension, Alzheimer's disease or dementia.
 3. The method of claim 1,wherein the cytotoxic agent is a radioisotope, and wherein theradioisotope is either ¹³¹I or ⁹⁰Y.
 4. The method of claim 1, whereinthe biospecific drug conjugate kills the senescent cell.